DESCRIPTION: (Applicant's Abstract) The long term objective of this project is to understand at a molecular level the detailed permeation and gating properties of mammalian inositol 1,4,5-trisphosphate receptors (InsP3R) and the mechanisms and kinetics of their regulation. InsP3Rs are ligand-gated Ca2+ channels expressed in endoplasmic reticulum (ER) membranes in possibly all cell types, releasing stored Ca2+ into the cytoplasm upon binding InsP3. InsP3R-mediated [Ca2+]i signals regulate diverse cell physiological processes, and are precisely controlled in both time and space, being manifested as oscillations and waves, and exhibiting unusual features including quantal release. The InsP3Rs are a family of proteins with diverse expression, suggesting that cells require distinct InsP3Rs to regulate their functions. However, the functional correlates of this diversity are largely unknown, in part because the InsP3R in its normal ER membrane environment has not been accessible for patch clamping, and the channel properties of recombinant channels have rarely been studied. A major limitation in understanding [Ca2+]i signalling has been the lack of precise data on the effects of modulators, including Ca2+, InsP3 and accessory proteins, on the permeation and gating properties of the different mammalian InsP3R channel isoforms. Furthermore, the lack of functional expression systems has resulted in an absence of data regarding the specific molecular determinants of the structural bases of InsP3R permeation, gating and regulation. We have now overcome these limitations by developing a novel nuclear patch clamp protocol for studying the single-channel properties of the InsP3R in its native ER membrane. Furthermore, we have also developed methodology for the successful expression and functional single channel measurements of different mammalian InsP3R isoforms. These novel developments now enable detailed investigations of the distinct mammalian InsP3R channels and the application of molecular biological approaches to address the structural bases of gating, permeation, regulation and subcellular localization. The specific aims of this proposal are to determine the single-channel properties of cloned mammalian InsP3R isoforms, by characterizing the permeation and gating properties of recombinant rat isoforms expressed in Xenopus oocyte nuclear membranes, and comparing their regulation. We will define the molecular determinants of InsP3R channel permeation, gating and regulation, by determining the structural basis of InsP3R inactivation, and defining sequences in the InsP3Rs involved in cytoplasmic Ca2+ regulation of channel gating and ion permeation. Finally, we will determine the mechanisms of heterogeneous subcellular localizations of InsP3R isoforms by defining their mobilities in the ER membrane, and the effects of cell activation and other pathways on InsP3R mobility, 1ocalization and clustering using recombinant rat GFP-InsP3R fusion proteins.